The B cell antigen receptor (BCR) serves dual, interrelated functions in B cell activation. The first is to initiate signal cascades that result in the transcription of a variety of genes associated with B cell activation. The second is to traffic bound antigen into the cell to deliver the antigen into intracellular compartments where the antigen is proteolytically cleaved and the resulting peptides are assembled into MHC class II-peptide complexes. The MHC class II-peptide complexes are expressed on the B cell surface for recognition by helper T cells that will as a consequence be activated to provide essential growth and differentiation factors to the B cell. The signaling and antigen transport functions of the BCR are interdependent in that the BCR signaling is necessary for the correct and rapid targeting of the antigen. In addition to the transport functions of the BCR, the internalization of the BCR may play an important role in down regulating signaling by removing activated receptors from the cell surface. At present it is not known if the antigen targeting and BCR down regulation are one in the same or separable processes. In addition, our current evidence indicates that the BCR enters the class II-containing MVBs as an intact, and presumably signaling competent, receptor. However, at present it is an open question as to whether the BCR signals intracellularly and if so what role intracellular signaling might play in B cell activation. The long term goal of this program is to gain a better understanding of the molecular mechanisms that underlie the correct targeting of the BCR to the class II-containing MVBs including determining where in the pathway discrete signaling events occur and to further define the role of intracellular signaling on B cell activation with particular focus on the interaction of the BCR with the intracellular Toll like receptors (TLRs).[unreadable] [unreadable] Our earlier findings investigating the effect of the expression of the Epstein Barr Virus latent membrane protein 2A (LMP2A) on BCR signaling and trafficking provided evidence that a specific subset of BCR signals were required for correct targeting. We observed that expression of the wild type LMP2A blocked both BCR signaling and trafficking but that a mutation in a tyrosine in the cytoplasmic domain of LMP2A (Y112) allowed the initiation of BCR signaling but still blocked BCR trafficking to the class II-containing MVBs. The ability of the mutant Y112-LMP2A to differentially affect BCR signaling and antigen trafficking provided a tool by which the signaling requirements for BCR internalization and trafficking could be dissected. [unreadable] [unreadable] A detailed analysis of B cell lines expressing Y112-LMP2A revealed that Y112-LMP2A expression completely blocked the BCR-induced activation of phospholipase D (PLD). PLD is a signal-transduction-activated enzyme that hydrolyzes phosphatidylcholine to generate the second messenger phosphatic acid and choline. PLD has been implicated in controlling a wide variety of cellular processes including the endocytosis and vesicular trafficking of cellular receptors. We showed that blocking PLD activity by either expressing Y112-LMP2A, treating cells with the PLD inhibitor, 1-butanol, or reducing PLD expression by siRNA, did not block antigen-induced internalization of the BCR but did block its correct trafficking to the class II-containing compartment. Thus, the activity of PLD did not appear to be required for endocytosis of the BCR from the plasma membrane but was required for subsequent steps in the BCRs trafficking pathway. [unreadable] [unreadable] We have begun to map the subcellular location in which components of the BCR signaling pathway are activated by confocal microscopy using antibodies specific for the phosphorylated forms of signaling components including Syk, p38, ERK and JNK. Our results indicated that components of the BCR signaling pathway are activated sequentially and in defined subcellular locations. We observed that pSyk appeared on the plasma membrane immediately following BCR crosslinking, whereas the phosphorylated forms of p38, ERK and JNK were not detected until the BCR had internalized from the plasma membrane and trafficked to the MVBs. Indeed, blocking BCR internalization by dansylcadaverine blocked the phosphorylation of both ERK and JNK. The internalized BCR colocalized in LAMP-positive MVBs with pERK and pJNK. [unreadable] [unreadable] The observations described above, that the BCR continues to signal as it is internalized to the MVBs suggested that intracellular BCR signaling may provide a function in B cell activation. BCR signaling is regulated and fine tuned by an array of coreceptors many of which are present on the cell surface where they presumably interact with the BCR. However, members of one family of receptors, the TLRs, that have been shown to influence BCR signaling are located in intracellular endosomes and not on the plasma membrane. One endosomal TLR, TLR9, that recognizes unmethylated CpG-DNA motifs has been shown to enhance BCR signaling resulting in hyperactivation of B cells to proliferate and differentiate into antibody secreting plasma cells. Recent evidence indicates that synergistic signaling between the BCR and TLR9 underlies the production of self-reactive antibodies in systemic autoimmune diseases and partly explains the preponderance of auto-antibodies that react with DNA-containing antigens. [unreadable] [unreadable] Both the TLR9- and BCR-signaling pathways ultimately result in activation of the p38, JNK and NFKB. Although the BCR and TLR9 initiate signaling from two spatially distinct sites, the BCR from the plasma membrane, and TLR9 from endosomes, our observation that the BCR-induced activation of p38 and JNK occurs at the MVBs suggest that the MVBs may be a site of synergistic signaling between the BCR and TLR9 in response to DNA-containing antigens. Using confocal microscopy and antibodies specific for the BCR, TLR9, p38 and pJNK we showed that following the binding and internalization of DNA-containing antigens, the BCR signals for the recruitment of TLR9 from multiple small endosomes to a large LAMP1-positive MVB into which the BCR trafficks antigen and where synergistic signaling through to p38 and JNK occurs. The recruitment of TLR9 to the MVBs was by a dynein-mediated, microtubule-network dependent process and was necessary for B cell hyper-responses to DNA-containing antigens. Of considerable interest was the observation that recruitment of TLR9 was dependent on the BCRs activation of PLD. Thus, PLD plays a role in both the correct targeting of the BCR to the MVBs and the recruitment of TLR9 to the same MVBs. The BCRs PLD-activation dependent recruitment of the TLR9 to the MVB represents a novel mechanism underlying the hyper-response of B cells to DNA-containing antigens.